The Angiotensin Metabolite His-Leu Is a Strong Copper Chelator Forming Highly Redox Active Species

His-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet–visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 μM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu.

Table S1.Potential and current values of redox processes obtained by cyclic voltammetry for 0.45 mM Cu(II) and ligands, His-Leu, histidine (His) and histamine (Hstm), at concentrations given in the Table .The measurements for 0.50 mM His-Leu and 2.5 mM ligands were repeated at least three times and the respective standard deviation values at the last significant digit are in the parenthesis.

Figure S1 .
Figure S1.UV−Vis titrations of 2.75 mM His-Leu/2.50 mM Cu(II) (A) and 5.00 mM His-Leu/2.50 mM Cu(II) (B) with NaOH codded with rainbow colours from red (pH 2) to violet (pH 12) as provided in the figure legend.

Figure S2 .
Figure S2.UV−Vis (A, C, E) and CD (B, D, F) spectra of the titrations of 2.75 mM His-Leu/ 2.50 mM Cu(II) with NaOH divided into three pH subranges, pH 2.4−6.5 (A, B), pH 6.5−8.6 (C, D), and pH 8.6−12.2(E, F) codded with rainbow colours as provided in the figure legends.The arrows represent the direction of spectral changes in the given pH subrange.

Figure S3 .
Figure S3.UV−Vis (A, C, E) and CD (B, D, F) spectra of the titrations of 5.00 mM His-Leu/ 2.50 mM Cu(II) with NaOH divided into three pH subranges, pH 2.0−4.3 (A, B), pH 4.3−7.5 (C, D), and pH 7.5−11.6(E, F) codded with rainbow colours as provided in the figure legends.The arrows represent the direction of spectral changes in the given pH subrange.

Figure S6 .
Figure S6.UV−Vis (A) and CD (B) spectra of the titration of 0.6 mM His-Leu/0.5 mM Cu(II) with His-Leu at pH 7.4.The selected final concentrations of the peptide in the cuvette for the given spectrum are provided in the legend.The arrows represent the direction of spectral changes during the experiment.(C) The comparison of CuL2 molar fraction with the spectral changes (A670 and θ690−710) registered during the titration.

Figure S7 .
Figure S7.Concentration dependence of rt−EPR spectra recorded at 24 C for solutions containing Cu(II) and His-Leu at pH 7.4 and the 1.0:1.2molar ratio, and Cu(II) concentrations of 10.0, 7.5, 5.0, 2.5, 1.0, and 0.5 mM codded with the gradient colours from dark green (10.0 mM Cu(II)) to orange (0.5 mM Cu(II)).Inset shows concentration dependence of signal amplitude, obtained as a difference of integrated 1 st derivative amplitudes integrated over the shaded spectral ranges, and its linear fit (R 2 = 0.994).

Figure S8 .
FigureS8.The rt−EPR spectra simulated (solid lines) for individual complex species using EasySpin, compared with their experimental counterparts (dashed lines).

Figure S10 .
Figure S10.CV (A) and DPV (B) curves registered after the addition of 0.45 mM Cu(NO3)2 to 0.1 M KNO3 and pH adjusting to 7.4.The arrows represent the direction of potential change.

Figure S11 .
Figure S11.Comparison between the Cu(II) molar fractions of bis complexes at conditions of electrochemical measurements and CV (circles) or DPV (diamonds) potential values.The scale for the right Y-axis was omitted as it represents several Y-axis for different redox processes, which cover various ranges of E values.

Figure S12 .
Figure S12.UV-vis spectra recorded during the incubation of Cu(II)/His-Leu with ascorbate at four reagent concentrations, 0.5 mM Cu(II)/0.6 mM His-Leu/1 mM AscH -(A), 0.5 mM Cu(II)/2.5 mM His-Leu/1 mM AscH -(B), 0.5 mM Cu(II)/0.6 mM His-Leu/5 mM AscH -(C), and 0.5 mM Cu(II)/2.5 mM His-Leu/5 mM AscH -(D).All incubations were performed for 24 hours in 50 mM HEPES pH 7.4.The dashed spectra refer to signals for Cu(II)/His-Leu complexes without AscH -.The spectra after the AscH - addition are codded with rainbow colors from dark red to blue.The insets are for zooming the changes near the d-d region.The arrows indicate the direction of the band changes during the reaction.

Figure S13 .
Figure S13.The changes in absorbance for d-d bands (A, C) and for the band at around 400 nm (B, D) during 24-hour kinetics recorded for 0.5 mM Cu(II)/0.6 mM His-Leu (A, B) and 0.5 mM Cu(II)/2.5 mM His-Leu (C, D) after the addition of 1 mM AscH -(black points for the reaction in 50 mM HEPES pH 7.4 and blue points for the reaction in 50 mM phosphate pH 7.4) or the addition of 5 mM AscH -(red points for the reaction in 50 mM HEPES pH 7.4).The band around 400 nm comprises two maxima at 390 nm and 425 nm, whose intensity increases proportionally to each other, as shown in B and D, where dot symbols representing absorbance at 390 nm are overlapped by triangles representing absorbance at 425 nm.

Figure S15 .
Figure S15.(A) UV-vis spectra recorded during the incubation of 0.5 mM Cu(II)/0.6 mM His-Leu with 1 mM AscH -in 50 mM phosphate buffer pH 7.4 for 24 h.The dashed spectrum refers to signals for Cu(II)/His-Leu complexes in phosphate without AscH -.The spectra after the AscH -addition are codded with rainbow colors from dark red to blue.The insets are for zooming the changes near the d-d and region.The arrows indicate the direction of the band changes during the reaction.(B) The comparison of the UV-vis spectra of 0.5 mM Cu(II)/0.6 mM His-Leu in 50 mM HEPES pH 7.4 (solid lines) and in 50 mM phosphates (dotted lines) without ascorbate (black lines) and after 2-hour incubation with ascorbate (red lines).

Figure S17 .
Figure S17.Cu(II) molar fraction for bis complexes of His-Leu and Cu(II) ions calculated based on the potentiometric constants for the His−Leu/Cu(II) molar ratio range 1−100 and the Cu(II) concentrations from 1 μM to 1 mM at pH 7.4.For clarity, the inset represents the curves in the narrowed His-Leu/Cu(II) molar ratio range 1−10.

Table S2 .
Potential and current values of redox processes obtained by differential pulse voltammetry for 0.45 mM Cu(II) and ligands, His-Leu, histidine (His) and histamine (Hstm), at concentrations given in the Table.The measurements for 0.50 mM His-Leu and 2.5 mM ligands were repeated at least three times and the respective standard deviation values at the last significant digit are in the parenthesis.
*the low intensity signal noticed by DPV and CV measurements at v = 10 mV/s

Table S3 .
Cu(II) species distributions for Cu(II) complexes of His-Leu calculated for concentrations used in electrochemical measurements and pH 7.4

Table S4 .
Cu(II) species distributions for Cu(II), His-Leu, histamine (Hstm), histidine (His) concentrations and pH 7.4 used in the ascorbate oxidation assay a calculated based on literature potentiometric results 1 b calculated based on literature potentiometric results2